Membrane tissue treatment compositions, membrane tissue treatment reagents, tissue repair materials, their preparation methods and applications

Abstract

This application relates to the field of tissue engineering and medical biomaterials, and particularly to a membrane tissue treatment composition, a membrane tissue treatment reagent, a tissue repair material, its preparation method, and its application. The membrane tissue treatment composition comprises tea polyphenols and gallic acid in a molar ratio of 1:(2-8). The combination of tea polyphenols and gallic acid exhibits a synergistic effect in improving the performance of the repair material.

Description

Technical Field

[0001] This application relates to the field of tissue engineering and medical biomaterials, and in particular to a membrane tissue treatment composition, membrane tissue treatment reagent, tissue repair material, preparation method and application thereof. Background Technology

[0002] Oral repair membranes are widely used in oral medicine fields such as periodontology, dental implantology, and alveolar surgery. An oral repair membrane is a biocompatible material that is surgically placed between the oral soft tissue and the bone defect area to create a biological barrier. This creates a relatively closed bone regeneration environment, selectively blocking fast-migrating fibroblasts and epithelial cells from entering the bone defect area, while not hindering natural wound healing.

[0003] Oral repair membranes can be categorized based on their material source, including collagen membranes, metal membranes, synthetic membranes, and allogeneic bone membranes. They can also be categorized based on their biodegradability, into absorbable and non-absorbable membranes. Non-absorbable membranes require a second surgery for removal. While non-collagen absorbable membranes do not require a second surgery, they can cause inflammation due to acidic degradation products. Therefore, considering all factors, collagen membranes offer the most comprehensive advantages.

[0004] Common collagen membranes are derived from decellularized animal tissue membranes. After processing, decellularized animal tissue membranes form a natural bilayer collagen structure. Under an electron microscope, one layer of collagen fibers is densely packed, while the other layer is loosely packed. This unique bilayer structure has significant clinical implications. The dense side provides a natural plane for epithelial cell migration, facilitating rapid epithelialization. The loose side fully retains the skin's natural three-dimensional structure, possessing suitable pore size and porosity, which promotes cell migration and growth. It provides a good scaffold for host cell growth and rapid vascularization, regulating, guiding, and promoting cell ingrowth and vascularization, thereby completing the repair and reconstruction of tissue defects.

[0005] Currently, decellularized animal tissue membranes from various tissues have been researched and applied. Commercialized tissue-engineered skin, cartilage, and other products have officially entered clinical use. Clinical applications of tissue-engineered bone, tendons, skeletal muscle, cornea, mucous membranes, blood vessels, bladder, pancreas, reproductive organs, kidneys, and liver are also beginning and have achieved some therapeutic effects. Decellularized animal tissue membranes can serve as the foundation for tissue engineering, offering significant advantages over synthetic materials. They not only retain the natural three-dimensional structure and extracellular matrix, possessing non-immunogenicity, biodegradability, sealing properties, non-toxicity, non-carcinogenicity, good biocompatibility, and mechanical properties, but also retain basic fibroblast growth factor, making them considered ideal renewable scaffold materials.

[0006] In the preparation of decellularized animal tissue membranes, commonly used decellularization methods include physical methods (freeze-thaw, pressurization, ultrasound, etc.), chemical methods (acids, alkalis, hypotonic and hypertonic solutions, nonionic detergents, ionic detergents, amphoteric detergents, metal ion chelating agents, etc.), enzymatic methods (nucleases, trypsin, lipases, etc.), and combinations of the above methods. Different decellularization methods result in decellularized animal tissue membranes with varying mechanical properties. Traditional decellularization methods are described in documents such as CN114191613A, CN104083803A, and CN118236556A. Improving the mechanical properties of decellularized animal tissue membranes is the primary goal of the decellularization process.

[0007] In view of the above, this application is hereby submitted. Summary of the Invention

[0008] This application provides one or more embodiments of a membrane tissue treatment composition, a membrane tissue treatment reagent, a tissue repair material, a preparation method thereof, and its application. The technical solutions include the following:

[0009] One or more embodiments of this application provide a membrane tissue treatment composition comprising tea polyphenols and gallic acid in a molar ratio of 1:(2-8).

[0010] In some embodiments of this application, the membrane tissue treatment composition further includes a high molecular weight polysaccharide compound, which includes one or more of ethyl cellulose and hydroxypropyl cellulose;

[0011] The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is 1:(2-8):(3-5).

[0012] One or more embodiments of this application also provide a membrane tissue treatment reagent, the membrane tissue treatment reagent comprising tea polyphenols, gallic acid and a solvent;

[0013] The molar ratio of tea polyphenols to gallic acid is 1:(2-8).

[0014] In some embodiments of this application, the total concentration of the tea polyphenols and the gallic acid is 0.01M to 1M.

[0015] In some embodiments of this application, the membrane tissue treatment reagent further includes a high molecular weight polysaccharide compound, which includes one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenol, the gallic acid and the high molecular weight polysaccharide compound is 1:(2-8):(3-5).

[0016] In some embodiments of this application, the total concentration of the tea polyphenols, the gallic acid, and the high molecular weight polysaccharide compound is 0.01M to 1M.

[0017] In some embodiments of this application, the solvent includes PBS buffer.

[0018] In some embodiments of this application, the PBS buffer comprises 0.1 mol / L to 0.3 mol / L disodium hydrogen phosphate and 0.1 mol / L to 0.3 mol / L sodium dihydrogen phosphate, with a pH of 6.5 to 7.5.

[0019] One or more embodiments of this application also provide a method for preparing a tissue repair material, the method comprising the step of decellularizing the biomembrane material using the membrane tissue treatment reagent described above.

[0020] In some embodiments of this application, the decellularization treatment conditions include: being carried out under shaking conditions, the treatment being performed 2 to 5 times, and each treatment lasting 30 to 90 minutes.

[0021] In some embodiments of this application, the preparation method includes multiple decellularization treatment stages, in which the membrane tissue treatment reagent is used to decellularize the biomembrane material in one of the decellularization treatment stages.

[0022] In some embodiments of this application, the preparation method includes a first decellularization treatment stage and a second decellularization treatment stage, wherein the biomembrane material is decellularized using the membrane tissue treatment reagent in the second decellularization treatment stage.

[0023] In some embodiments of this application, the first decellularization treatment stage includes: placing the biofilm material sequentially in a hypertonic solution and a hypotonic solution for decellularization treatment.

[0024] In some embodiments of this application, the first decellularization treatment stage satisfies one or more of the following conditions:

[0025] 1) The hypertonic solution comprises a 0.01M to 1M sodium chloride solution with 0.01M to 1M alkali or acid added; optionally, the alkali comprises one or more of sodium hydroxide and potassium hydroxide; optionally, the acid comprises one or more of hydrochloric acid and acetic acid.

[0026] 2) The hypotonic solution includes water;

[0027] 3) The first decellularization treatment step is repeated 2 to 5 times; and,

[0028] 4) The process is carried out under shaking conditions, with the shaking time in the hypertonic solution and the hypotonic solution being 30 min to 90 min each independently.

[0029] In some embodiments of this application, the biofilm material is treated as follows: removing attached fat, connective tissue and edge-damaged tissue, defatting and inactivating viruses.

[0030] In some embodiments of this application, degreasing satisfies one or more of the following conditions:

[0031] (I) The degreasing agents used include one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane, and ethyl acetate; and,

[0032] (II) Perform the degreasing treatment under shaking conditions, 2 to 4 times, each time for 2 to 10 hours. After each degreasing treatment, replace with fresh degreasing reagent before the next degreasing treatment.

[0033] In some embodiments of this application, virus inactivation is performed using chemical methods.

[0034] In some embodiments of this application, the chemical method satisfies one or more of the following conditions:

[0035] (I) The inactivating reagent used includes one or more of an acid, a base, and an alcohol; optionally, the base includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide, and ammonia water; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid, and acetic acid; the alcohol includes one or more of ethanol, propanol, isopropanol, and methanol; and,

[0036] (ii) The inactivation of the virus is carried out under static conditions for 1 to 3 hours.

[0037] In some embodiments of this application, after decellularization, the resulting tissue repair material is further frozen, sectioned, and sterilized.

[0038] In some embodiments of this application, the freezing conditions include: freeze drying is performed by cooling to -80°C to -20°C at a rate of 5°C / min to 12°C / min, and maintaining the temperature at -20°C to -10°C for 10h to 16h.

[0039] In some embodiments of this application, the thickness of the sliced ​​control film is 0.1 mm to 1 mm.

[0040] In some embodiments of this application, sterilization is performed using physical sterilization or chemical sterilization.

[0041] In some embodiments of this application, the biofilm material satisfies one or more of the following conditions:

[0042] (i) The biomembrane material is derived from the dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valves, small intestine, or basement membrane; and,

[0043] (ii) The biofilm material is derived from pigs, cattle or sheep.

[0044] One or more embodiments of this application also provide a tissue repair material, prepared by the method described above.

[0045] One or more embodiments of this application also provide a method for repairing bone defects, the method comprising the step of repairing the bone defect area using the tissue repair material described above.

[0046] In some embodiments of this application, the bone defect area is located in the oral cavity.

[0047] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 Tissue section images of the tissue repair material prepared using the product of the first decellularization stage and the tissue repair material prepared using the product of the second decellularization stage in Example 1;

[0050] Figure 2 The images show the microstructure of the tissue repair material prepared using the product of the first decellularization stage and the tissue repair material prepared using the product of the second decellularization stage in Example 1.

[0051] Figure 3 The images show animal experiments demonstrating the effectiveness of the tissue repair materials prepared using the products of the first and second decellularization stages in Example 1. Detailed Implementation

[0052] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0054] the term

[0055] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0056] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0057] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0058] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0059] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0060] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0061] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0063] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0064] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0065] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0066] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0067] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0068] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0069] Methods for decellularizing biological scaffolds are mainly divided into physical, chemical, and enzymatic methods. Regardless of the method, all decellularization methods can disrupt the structure and composition of the extracellular matrix (ECM). Due to tissue-specific factors such as cell density, matrix density, and geometric factors including tissue thickness and shape, the optimal method for decellularization varies from tissue to tissue and organ to organ. Because cellular residues cannot be completely removed, the decellularization process may cause some damage to the matrix. The effectiveness of cell removal from tissue depends on the tissue source and the decellularization method used. Each method affects the biochemical composition, tissue ultrastructure, and mechanical properties of the remaining ECM, thereby affecting the body's response to the material and hindering tissue repair and regeneration. To address the shortcomings of existing technologies, one objective of this application is to provide a method for preparing tissue repair materials that ensures thorough decellularization, produces materials with antioxidant and anti-chronic inflammatory effects, and exhibits good biocompatibility. This is beneficial for tissue repair and regeneration. Based on the removal of immunogenic substances from the material, innovative anti-inflammatory and antioxidant agents, including tea polyphenols, gallic acid, and ethyl cellulose, are introduced to promote tissue healing, induce bone regeneration, and enhance mechanical properties. This significantly improves the clinical operation of existing products and enhances their effectiveness.

[0070] Tea polyphenols, due to their antioxidant and antimicrobial properties, can be used as a biological additive in food packaging materials to reduce oxidation and prevent food spoilage and contamination by infectious pathogens. Simultaneously, tea polyphenols also play an important role in the treatment of chronic inflammation.

[0071] Gallic acid (GA), a natural polyphenol commonly found in plants, possesses strong antioxidant and antibacterial properties. Ethyl cellulose is a high-molecular-weight polysaccharide compound. Using one or more of tea polyphenols, gallic acid, and ethyl cellulose to prepare a solution can enhance the antioxidant and antibacterial effects of decellularized biological materials, exhibiting good biocompatibility. This is beneficial for tissue repair and regeneration.

[0072] The inventors of this application also unexpectedly discovered that the combined use of tea polyphenols and gallic acid solutions has a synergistic effect, which is better than the effect of using them alone. Furthermore, the complex solution system formed by ethyl cellulose with tea polyphenols and gallic acid is superior to other similar alternatives.

[0073] After treatment with the above reagents, the membranes are dried using a thickness-shaving device, which allows for more precise control of the product's thickness uniformity. This enables the preparation of products with the required thickness according to clinical market demands, significantly improving the industrial utilization rate of animal membranes, reducing production costs, and increasing production capacity. Furthermore, the shaving process preserves the natural tissue fiber structure, makes the material softer, and better conforms to soft tissue surfaces, facilitating clinical use and enhancing efficacy.

[0074] A first aspect of this application provides a membrane tissue treatment composition comprising tea polyphenols and gallic acid in a molar ratio of 1:(2-8). The molar ratio of tea polyphenols to gallic acid is, for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, or 1:8.

[0075] In some examples, the membrane tissue treatment composition further includes a high molecular weight polysaccharide compound, which includes one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenols, the gallic acid, and the high molecular weight polysaccharide compound is 1:(2-8):(3-5). For example, the molar ratio of the tea polyphenols, the gallic acid, and the high molecular weight polysaccharide compound is 1:2:3, 1:2.5:3, 1:3:3, 1:3.5:3, 1:4:3, 1:4.5:3, 1:5:3, 1:5.5:3, 1:6:3, 1:6.5:3, 1:7:3, 1:7.5:3, 1:8:3, 1:2:4, 1:2.5:4, 1:3:4, 1:3.5 ... :4:4, 1:4.5:4, 1:5:4, 1:5.5:4, 1:6:4, 1:6.5:4, 1:7:4, 1:7.5:4, 1:8:4, 1:2:5, 1:2.5:5, 1:3:5, 1:3.5:5, 1:4:5, 1:4.5:5, 1:5:5, 1:5.5:5, 1:6:5, 1:6.5:5, 1:7:5, 1:7.5:5, 1:8:5.

[0076] A second aspect of this application provides a membrane tissue treatment reagent, comprising tea polyphenols, gallic acid, and a solvent; the molar ratio of the tea polyphenols to the gallic acid is 1:(2-8), for example 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, or 1:8. Optionally, the total concentration of the tea polyphenols and the gallic acid is 0.01M to 1M, for example 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1M.

[0077] In some examples, the membrane tissue treatment reagent further includes a high molecular weight polysaccharide compound, which includes one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenols, the gallic acid, and the high molecular weight polysaccharide compound is 1:(2-8):(3-5), for example 1:2:3, 1:2.5:3, 1:3:3, 1:3.5:3, 1:4:3, 1:4.5:3, 1:5:3, 1:5.5:3, 1:6:3, 1:6.5:3, 1:7:3, 1:7. 5:3, 1:8:3, 1:2:4, 1:2.5:4, 1:3:4, 1:3.5:4, 1:4:4, 1:4.5:4, 1:5:4, 1:5.5:4, 1:6:4, 1:6.5:4, 1:7:4, 1:7.5:4, 1:8:4, 1:2:5, 1:2.5:5, 1:3:5, 1:3.5:5, 1:4:5, 1:4.5:5, 1:5:5, 1:5.5:5, 1:6:5, 1:6.5:5, 1:7:5, 1:7.5:5, 1:8:5. Optionally, the total concentration of the tea polyphenols, the gallic acid, and the high molecular weight polysaccharide compounds is 0.01M to 1M, for example, 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1M.

[0078] In some examples, the solvent includes PBS buffer. Optionally, the PBS buffer comprises 0.1 mol / L to 0.3 mol / L (e.g., 0.1, 0.15, 0.2, 0.25, 0.3 mol / L) of disodium hydrogen phosphate and 0.1 mol / L to 0.3 mol / L (e.g., 0.1, 0.15, 0.2, 0.25, 0.3 mol / L) of sodium dihydrogen phosphate, with a pH of 6.5 to 7.5 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5).

[0079] A third aspect of this application provides a method for preparing a tissue repair material, the method comprising the step of decellularizing the biomembrane material using the aforementioned membrane tissue treatment reagent.

[0080] In some examples, the decellularization treatment conditions include: treatment under shaking conditions, treatment times of 2 to 5 (e.g., 2, 3, 4, 5 times), and each treatment time of 30 to 90 minutes (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 minutes).

[0081] In some examples, the preparation method includes multiple decellularization stages, in one of which the membrane tissue treatment reagent is used to decellularize the biomembrane material.

[0082] In some examples, the preparation method includes a first decellularization treatment stage and a second decellularization treatment stage, wherein the biomembrane material is decellularized using the membrane tissue treatment reagent in the second decellularization treatment stage.

[0083] In some examples, the first decellularization treatment stage includes the step of sequentially placing the biofilm material in a hypertonic solution and a hypotonic solution for decellularization treatment. Optionally, the first decellularization treatment stage satisfies one or more of the following conditions:

[0084] 1) The hypertonic solution comprises a 0.01M to 1M (e.g., 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1M) sodium chloride solution with added 0.01M to 1M (e.g., 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1M) base or acid; optionally, the base comprises one or more of sodium hydroxide and potassium hydroxide; optionally, the acid comprises one or more of hydrochloric acid and acetic acid;

[0085] 2) The hypotonic solution includes water;

[0086] 3) The first decellularization treatment step is repeated 2 to 5 times (e.g., 2, 3, 4, 5 times); and,

[0087] 4) The shaking is carried out under shaking conditions, and the shaking time in the hypertonic solution and the hypotonic solution is independently 30 min to 90 min (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 min).

[0088] In some examples, the biofilm material is treated as follows: removal of attached fat, connective tissue and edge-damaged tissue, degreasing and virus inactivation.

[0089] This application does not specifically limit the degreasing steps in its embodiments. In some examples, degreasing satisfies one or more of the following conditions:

[0090] (I) The degreasing agents used include one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane, and ethyl acetate; and,

[0091] (II) Perform the degreasing treatment under shaking conditions, 2 to 4 times (e.g., 2, 3, 4, 5 times), each time for 2 to 10 hours (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 hours). After each degreasing treatment, replace with fresh degreasing reagent before the next degreasing treatment.

[0092] This application does not specifically limit the method of virus inactivation, including but not limited to chemical methods; in some examples, the chemical method satisfies one or more of the following conditions:

[0093] (I) The inactivating reagent used includes one or more of an acid, a base, and an alcohol; optionally, the base includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide, and ammonia water; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid, and acetic acid; the alcohol includes one or more of ethanol, propanol, isopropanol, and methanol; and,

[0094] Ⅱ) The inactivation is carried out under static conditions, and the inactivation time is 1h to 3h (e.g., 1, 1.5, 2, 2.5, 3h).

[0095] It is understood that, after decellularization, the resulting tissue repair material is frozen, sectioned, and sterilized in the embodiments of this application. The embodiments of this application do not particularly limit the specific steps of freezing, sectioning, and sterilization.

[0096] In some examples, the freezing conditions include: freeze drying at a rate of 5°C / min to 12°C / min (e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12°C / min) to -80°C to -20°C (e.g., -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20°C), and then holding at -20°C to -10°C (e.g., -20, -18, -16, -14, -12, -10) for 10h to 16h (e.g., 10, 11, 12, 13, 14, 15, 16h).

[0097] In some examples, the slice thickness is controlled to be 0.1 mm to 1 mm, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mm.

[0098] In some examples, sterilization is performed using physical sterilization methods. In other examples, sterilization is performed using chemical sterilization methods.

[0099] In some examples, the biofilm material satisfies one or more of the following conditions:

[0100] (i) The biomembrane material is derived from the dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valves, small intestine, or basement membrane; and,

[0101] (ii) The biofilm material is derived from pigs, cattle or sheep.

[0102] A fifth aspect of this application provides a tissue repair material prepared by the method for preparing tissue repair materials described in the fourth aspect.

[0103] A sixth aspect of this application provides a method for repairing bone defects, the method comprising the step of repairing the bone defect area using the tissue repair material described in the fifth aspect.

[0104] In some examples, the bone defect area is located in the oral cavity.

[0105] The embodiments of this application do not specifically limit the subjects to be repaired; they can be any animal that may have bone defects and needs repair. This can be a human or other non-human mammals. In this application, the term "mammal" primarily refers to warm-blooded vertebrate mammals, including but not limited to: cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats and mice), pigs, cattle, sheep, horses, and humans, preferably primates, and more preferably humans.

[0106] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0107] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0108] Example 1

[0109] Step 1, Pretreatment: Lay the bovine pericardium tissue flat on a plate, remove attached fat, connective tissue and damaged tissue at the edges, then wash with water until there is no blood, to obtain the biofilm material.

[0110] Step 2, Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for 6 hours to degrease. The solution is then changed, and the degreasing is repeated 3 times. The material is then washed with water until there is no odor. The organic reagent is propanol.

[0111] Step 3: Virus inactivation: Place the obtained biofilm material in a chemical reagent and let it stand for 2 hours to inactivate the virus. Wash it with water until there is no odor. The chemical reagent is a sodium hydroxide solution with a sodium hydroxide content of 0.1M.

[0112] Step 4: Decellularization

[0113] The first decellularization stage: The virus-inactivated biofilm material was placed in a decellularized hypertonic solution and shaken for 40 minutes, then transferred to a decellularized hypotonic solution and shaken for 60 minutes. This cycle was repeated 3 times. The decellularized hypertonic solution was an aqueous solution containing 0.5M sodium chloride and 1M sodium hydroxide, and the decellularized hypotonic solution was water.

[0114] The second decellularization stage used PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7).

[0115] Dissolve tea polyphenols, gallic acid, and ethyl cellulose to prepare a treatment reagent. The concentrations of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent are 0.02M, 0.04M, and 0.06M, respectively. Shake the biofilm material in the treatment reagent for 50 minutes. After shaking, replace with fresh treatment reagent. Repeat this cycle three times (i.e., shake once, then treat with fresh treatment reagent for the second time, and then treat with fresh treatment reagent for the third time). Wash with water until there is no odor.

[0116] Step 5, freeze drying: Flatten the biofilm material obtained in step 4, attach it to a plate, and put it directly into a freeze dryer for freeze drying. The freeze drying process involves cooling the material to -80°C at a rate of 8°C / min and maintaining it at -20°C for 16 hours. After freeze drying, the material is cut and packaged.

[0117] Step 6, Cutting: The freeze-dried biofilm is processed using a cutting device. The membrane thickness is precisely cut to 0.4mm to achieve uniform thickness of single-specification material.

[0118] Step 7: Sterilization: Chemical sterilization is performed using ethylene oxide to obtain tissue repair material.

[0119] Example 2

[0120] Step 1, Pretreatment: The porcine peritoneum tissue is laid flat on a plate, and attached fat, connective tissue and damaged tissue at the edges are removed. Then it is washed with water until there is no blood, and the biofilm material is obtained.

[0121] Step 2, Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for 4 hours to degrease. The solution is then changed, and the degreasing is repeated 3 times. The material is then washed with water until there is no odor. The organic reagent is ethanol.

[0122] Step 3: Virus inactivation: Place the obtained biofilm material in a chemical reagent and let it stand for 1 hour to inactivate it. Wash it with water until there is no odor. The chemical reagent is a sodium hydroxide solution with a sodium hydroxide content of 0.1M.

[0123] Step 4: Decellularization

[0124] The first decellularization stage: The virus-inactivated biofilm material was placed in a decellularized hypertonic solution and shaken for 30 minutes, then transferred to a decellularized hypotonic solution and shaken for 60 minutes. This cycle was repeated 3 times. The decellularized hypertonic solution was an aqueous solution containing 0.5M sodium chloride and 1M sodium hydroxide, and the decellularized hypotonic solution was water.

[0125] The second decellularization stage: Tea polyphenols, gallic acid, and ethyl cellulose were dissolved in PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) to prepare the treatment reagent. The concentrations of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent were 0.02 M, 0.04 M, and 0.1 M, respectively. The biofilm material was shaken in the treatment reagent for 50 min. After shaking, the reagent was replaced with fresh reagent. This cycle was repeated 3 times, followed by washing with water until no odor remained.

[0126] Step 5, freeze drying: Flatten the biofilm material obtained in step 4, attach it to a plate, and put it directly into a freeze dryer. Freeze drying is carried out by cooling the material to -80°C at a rate of 8°C / min and maintaining it at -20°C for 16 hours. After freeze drying, cut and package it.

[0127] Step 6, Cutting: The freeze-dried biofilm is processed using a cutting device. The membrane thickness is precisely cut to 0.3mm to achieve uniform thickness of single-specification material.

[0128] Step 7: Sterilization: Sterilize by irradiation to obtain tissue repair material.

[0129] Example 3

[0130] This embodiment is a variation of Embodiment 1, and the differences from Embodiment 1 are as follows:

[0131] Step 2, Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for 2 hours to degrease. The solution is then changed, and the degreasing is repeated 4 times. The material is then washed with water until there is no odor. The organic reagent is propanol.

[0132] Step 3: Virus inactivation: Place the obtained biofilm material in hydrochloric acid and let it stand for 1 hour to inactivate it, then wash it with water until there is no odor.

[0133] Step 4: Decellularization

[0134] The first decellularization stage: The virus-inactivated biofilm material was placed in a decellularized hypertonic solution and shaken for 50 minutes, then transferred to a decellularized hypotonic solution and shaken for 30 minutes. This cycle was repeated 5 times. The decellularized hypertonic solution was an aqueous solution containing 2M sodium chloride and 0.01M sodium hydroxide, and the decellularized hypotonic solution was water.

[0135] The second decellularization stage: Tea polyphenols, gallic acid, and ethyl cellulose were dissolved in PBS buffer solution (containing 0.1 mol / L disodium hydrogen phosphate and 0.3 mol / L sodium dihydrogen phosphate, pH 6.5) to prepare the treatment reagent. The molar ratio of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent was 1:2:5, and the total concentration of tea polyphenols, gallic acid, and ethyl cellulose was 0.01 M. The biofilm material was shaken in the treatment reagent for 30 min. After shaking, the treatment reagent was replaced with fresh reagent. This cycle was repeated 5 times, followed by washing with water until no odor was detected.

[0136] Step 5, freeze drying: Flatten the biofilm material obtained in step 4, attach it to a plate, and put it directly into a freeze dryer for freeze drying. The freeze drying process involves cooling the material to -20°C at a rate of 5°C / min and maintaining it at -10°C for 10 hours. After freeze drying, the material is cut and packaged.

[0137] Step 6, Cutting: The freeze-dried biofilm is processed using a cutting device. The membrane thickness is precisely cut to 0.1mm to achieve uniform thickness of single-specification material.

[0138] The rest are the same as in Example 1.

[0139] Example 4

[0140] This embodiment is a variation of Embodiment 1, and the differences from Embodiment 1 are as follows:

[0141] Step 2, Degreasing: Place the obtained biofilm material in ethyl acetate and shake to degrease for 10 hours, then change the solution. Repeat the degreasing process 4 times, and wash with water until there is no odor.

[0142] Step 3: Virus inactivation: Place the obtained biofilm material in propanol and let it stand for 3 hours to inactivate the virus, then wash with water until there is no odor.

[0143] Step 4: Decellularization

[0144] First decellularization stage: The virus-inactivated biofilm material was placed in a decellularized hypertonic solution and shaken for 90 minutes, then transferred to a decellularized hypotonic solution and shaken for 90 minutes. This cycle was repeated twice. The decellularized hypertonic solution was an aqueous solution containing 5M sodium chloride and 0.5M sodium hydroxide, and the decellularized hypotonic solution was water.

[0145] The second decellularization stage: Tea polyphenols, gallic acid, and ethyl cellulose were dissolved in PBS buffer solution (containing 0.3 mol / L disodium hydrogen phosphate and 0.1 mol / L sodium dihydrogen phosphate, pH 7.5) to prepare the treatment reagent. The molar ratio of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent was 1:8:3, and the total concentration of tea polyphenols, gallic acid, and ethyl cellulose was 1M. The biofilm material was shaken in the treatment reagent for 50 min, and then replaced with fresh treatment reagent. This cycle was repeated 3 times, followed by washing with water until no odor was detected.

[0146] Step 5, freeze drying: Flatten the biofilm material obtained in step 4, attach it to a plate, and put it directly into a freeze dryer for freeze drying. The freeze drying process involves cooling the material to -16℃ at a rate of 12℃ / min and maintaining it at -15℃ for 14 hours. After freeze drying, the material is cut and packaged.

[0147] Step 6, Cutting: The freeze-dried biofilm is processed using a cutting device. The membrane thickness is precisely cut to 1mm to achieve uniform thickness of single-specification material.

[0148] The rest are the same as in Example 1.

[0149] Example 5

[0150] This embodiment is a variation of Embodiment 1, and the changes compared to Embodiment 1 include:

[0151] In step four, the second decellularization stage, ethyl cellulose was not added. Instead, tea polyphenols and gallic acid were dissolved in PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) to prepare the treatment reagent. The concentrations of tea polyphenols and gallic acid in the treatment reagent were 0.04 M and 0.08 M, respectively. The biofilm material was shaken in the solution for 50 min. After shaking, the treatment reagent was replaced with fresh reagent. This cycle was repeated three times, followed by washing with water until no odor remained.

[0152] The rest are the same as in Example 1.

[0153] Example 6

[0154] This embodiment is a variation of Embodiment 1, and the changes compared to Embodiment 1 include:

[0155] In step four, the second decellularization stage, ethyl cellulose was replaced with hydroxypropyl cellulose. The treatment reagent was prepared by dissolving tea polyphenols, gallic acid, and hydroxypropyl cellulose in PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7). The concentrations of tea polyphenols, gallic acid, and hydroxypropyl cellulose in the treatment reagent were 0.02 M, 0.04 M, and 0.06 M, respectively. The biofilm material was shaken in the treatment reagent for 50 min. After shaking, the reagent was replaced with fresh reagent. This cycle was repeated three times, followed by washing with water until no odor remained.

[0156] The rest are the same as in Example 1.

[0157] Example 7

[0158] This embodiment is a variation of Embodiment 1, and the changes compared to Embodiment 1 include:

[0159] In step four, the second decellularization stage, tea polyphenols, gallic acid, and ethyl cellulose were dissolved in PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) to prepare a treatment reagent. The concentrations of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent were 0.02 M, 0.04 M, and 0.04 M, respectively. The biofilm material was shaken in the treatment reagent for 50 min, and then replaced with fresh treatment reagent. This cycle was repeated three times, followed by washing with water until no odor remained.

[0160] The rest are the same as in Example 1.

[0161] Example 8

[0162] This embodiment is a variation of Embodiment 1, and the changes compared to Embodiment 1 include:

[0163] In step four, the second decellularization stage, tea polyphenols, gallic acid, and ethyl cellulose were dissolved in PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) to prepare the treatment reagent. The concentrations of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent were 0.02 M, 0.04 M, and 0.12 M, respectively. The biofilm material was shaken in the treatment reagent for 50 min, and then replaced with fresh treatment reagent. This cycle was repeated three times, followed by washing with water until no odor remained.

[0164] The rest are the same as in Example 1.

[0165] Example 9

[0166] This embodiment is a variation of Embodiment 1, and the changes compared to Embodiment 1 include:

[0167] In the second decellularization stage of step four, the molar concentration ratio of tea polyphenols, gallic acid, and ethyl cellulose is 1:2:3, and the total concentration is 1.2M.

[0168] The rest are the same as in Example 1.

[0169] Example 10

[0170] This embodiment is a variation of Embodiment 1, and the changes compared to Embodiment 1 include:

[0171] In the second decellularization stage of step four, the molar concentration ratio of tea polyphenols, gallic acid, and ethyl cellulose is 1:2:3, and the total concentration is 0.009M.

[0172] The rest are the same as in Example 1.

[0173] Comparative Example 1

[0174] This comparative example is a comparative example of Example 1, differing only in that: in step four, the second decellularization stage, PBS buffer solution was used to dissolve tea polyphenols and prepare the treatment reagent, with the concentration of tea polyphenols in the treatment reagent being 0.06M. All other aspects are the same as in Example 1.

[0175] Comparative Example 2

[0176] This comparative example is a comparative example of Example 1. The only difference from Example 1 is that in step four, the second decellularization stage, gallic acid was dissolved in PBS buffer solution to prepare the treatment reagent, and the concentration of gallic acid in the treatment reagent was 0.06M. All other aspects are the same as in Example 1.

[0177] Comparative Example 3

[0178] This comparative example is a comparative example of Example 1, and the only difference between it and Example 1 is that:

[0179] In step four, tea polyphenols are replaced by tannic acid. Specifically, tannic acid, gallic acid, and ethyl cellulose are dissolved in PBS buffer to prepare a treatment reagent. The concentrations of tannic acid, gallic acid, and ethyl cellulose in the treatment reagent are 0.02 M, 0.04 M, and 0.06 M, respectively. The rest is the same as in Example 1.

[0180] Comparative Example 4

[0181] This comparative example is a comparative example of Example 1, and the only difference between it and Example 1 is that:

[0182] In step four, the second decellularization stage, gallic acid is replaced by epigallocatechin gallate. Specifically, tea polyphenols, epigallocatechin gallate, and ethyl cellulose are dissolved in PBS buffer to prepare a treatment reagent. The concentrations of tea polyphenols, epigallocatechin gallate, and ethyl cellulose in the treatment reagent are 0.02 M, 0.04 M, and 0.06 M, respectively. All other steps are the same as in Example 1.

[0183] Comparative Example 5

[0184] This comparative example is a comparative example of Example 1, and the only difference between it and Example 1 is that:

[0185] In step four, the second decellularization stage, the molar ratio of tea polyphenols to gallic acid is 1:1. Specifically, tea polyphenols, gallic acid, and ethyl cellulose are dissolved in PBS buffer to prepare the treatment reagent. The concentrations of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent are 0.03M, 0.03M, and 0.06M, respectively. The rest is the same as in Example 1.

[0186] Comparative Example 6

[0187] This comparative example is a comparative example of Example 1, and the only difference between it and Example 1 is that:

[0188] In the second decellularization stage of step four, the molar ratio of tea polyphenols to gallic acid is 1:9. Specifically, tea polyphenols, gallic acid, and ethyl cellulose are dissolved in PBS buffer solution to prepare the treatment reagent. The concentrations of tea polyphenols, gallic acid, and ethyl cellulose in the treatment reagent are 0.01M, 0.09M, and 0.06M, respectively. The rest is the same as in Example 1.

[0189] Performance testing

[0190] Experiment 1: Histological sections

[0191] A tissue repair sample of at least 1.0 × 1.0 cm from Example 1 was examined using standard HE staining techniques. The results are as follows: Figure 1 As shown. Figure 1 In the image, the first row of three images shows tissue repair materials prepared using the products from the first decellularization stage, and the second row of three images shows tissue repair materials prepared using the products from the second decellularization stage.

[0192] Experiment 2: Microstructure Observation

[0193] The tissue repair material from Example 1, after being fully rehydrated for 5 minutes, was cut into pieces approximately 1 mm × 5 mm in size, stained, and observed using a transmission electron microscope. The results are shown below. Figure 2 , Figure 2The left image shows the tissue repair material prepared using the product of the first decellularization stage, and the right image shows the tissue repair material prepared using the product of the second decellularization stage. The results show that the tissue repair material of Example 1 has a complete natural collagen fiber structure, with a clear and distinct collagen fiber arrangement structure. A periodic striation / layered arrangement structure can be seen along the long axis of the collagen fibers.

[0194] Experiment 3: Mechanical Property Testing

[0195] The suture tear strength of the tissue repair material shall be tested in accordance with the provisions of YY 0500-2020;

[0196] The tensile strength and elongation at break of tissue repair materials were determined according to GB / T 3923.1-2013, Part 1 (strip method).

[0197] The results showed that each embodiment had stronger mechanical properties. The decellularization treatment reagent of this application can enhance the toughness of tissue repair materials. Furthermore, the combined use of tea polyphenols, gallic acid, and ethyl cellulose was more effective than the use of the three components alone, and the mechanical properties were more advantageous.

[0198] Table 1. Suture tear strength

[0199]

[0200] Table 1 shows tissue repair materials prepared using the products of the first decellularization stage ("after decellularization") and tissue repair materials prepared using the products of the second decellularization stage ("after solution treatment").

[0201] Table 2. Tensile breaking strength

[0202]

[0203]

[0204] Table 2 shows tissue repair materials prepared using the products of the first decellularization stage ("after decellularization") and tissue repair materials prepared using the products of the second decellularization stage ("after solution treatment").

[0205] Table 3. Elongation at break

[0206]

[0207]

[0208] Table 3 shows tissue repair materials prepared using the products of the first decellularization stage ("after decellularization") and tissue repair materials prepared using the products of the second decellularization stage ("after solution treatment").

[0209] Experiment 4: In vitro degradation detection

[0210] The tissue repair material samples from each example and comparative example were cut into uniform fragments and added to 10 mL of 0.1 M (pH 7.4) PBS buffer containing 50 CDU / mL L1 type collagenase. The mixture was then shaken at 37°C.

[0211] See results Figure 3 The results showed that the tissue repair material of Example 1 exhibited superior degradation performance and a longer in vitro degradation time. This barrier time was more advantageous in clinical use, effectively promoting tissue healing and leveraging the natural advantages of detachable tissue repair materials. Figure 3 The second row of the attached diagram shows tissue repair materials prepared using the products from the second decellularization stage. Figure 3 The first row of the attached figures shows tissue repair materials prepared using the products from the first decellularization stage.

[0212] Table 4. In vitro degradation

[0213]

[0214]

[0215] Table 4 shows tissue repair materials prepared using the products of the first decellularization stage ("after decellularization") and tissue repair materials prepared using the products of the second decellularization stage ("after solution treatment").

[0216] Experiment 5: Animal Experiment

[0217] The tissue repair material of Example 1 (i.e., the tissue repair material prepared using the product of the second decellularization stage) was tested to verify its effectiveness in filling canine tooth extraction sockets, and to verify the material's degradation performance and its healing performance on soft tissues.

[0218] The histological results from animal experiments show that: 2 weeks after implantation of the tissue repair material in Example 1, early vascularization occurred and early new bone formation had occurred under the collagen membrane. 8 weeks after implantation, the tissue repair material gradually degraded. 12-16 weeks after implantation, new bone continued to form, and the epithelium had covered the defect area.

[0219] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0220] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A membrane tissue treatment composition, characterized in that, The membrane tissue treatment composition comprises tea polyphenols and gallic acid in a molar ratio of 1:(2-8).

2. The membrane tissue treatment composition according to claim 1, characterized in that, The membrane tissue treatment composition further includes a high molecular weight polysaccharide compound, which includes one or more of ethyl cellulose and hydroxypropyl cellulose; The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is 1:(2-8):(3-5).

3. A membrane tissue treatment reagent, characterized in that, The membrane tissue treatment reagent includes tea polyphenols, gallic acid, and solvents; The molar ratio of tea polyphenols to gallic acid is 1:(2-8).

4. The membrane tissue treatment reagent according to claim 3, characterized in that, The total concentration of the tea polyphenols and the gallic acid is 0.01M to 1M.

5. The membrane tissue treatment reagent according to claim 4, characterized in that, The membrane tissue treatment reagent also includes a high molecular weight polysaccharide compound, which includes one or more of ethyl cellulose and hydroxypropyl cellulose; The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is 1:(2-8):(3-5).

6. The membrane tissue treatment reagent according to claim 5, characterized in that, The total concentration of the tea polyphenols, gallic acid, and high molecular weight polysaccharide compounds is 0.01M to 1M.

7. The membrane tissue treatment reagent according to any one of claims 3 to 6, characterized in that, The solvent includes PBS buffer.

8. The membrane tissue treatment reagent according to claim 7, characterized in that, The PBS buffer comprises 0.1 mol / L to 0.3 mol / L disodium hydrogen phosphate and 0.1 mol / L to 0.3 mol / L sodium dihydrogen phosphate, with a pH of 6.5 to 7.

5.

9. A method for preparing a tissue repair material, characterized in that, The preparation method includes the step of decellularizing the biomembrane material using the membrane tissue treatment reagent described in any one of claims 3 to 8.

10. The method for preparing the tissue repair material according to claim 9, characterized in that, The conditions for decellularization include: treatment under shaking conditions, repeated 2 to 5 times, each time for 30 to 90 minutes.

11. The method for preparing the tissue repair material according to claim 9, characterized in that, The preparation method includes multiple decellularization treatment stages, in which the membrane tissue treatment reagent is used to decellularize the biomembrane material in one of the decellularization treatment stages.

12. The method for preparing the tissue repair material according to claim 11, characterized in that, The preparation method includes a first decellularization treatment stage and a second decellularization treatment stage, wherein the biomembrane material is decellularized using the membrane tissue treatment reagent in the second decellularization treatment stage.

13. The method for preparing the tissue repair material according to claim 12, characterized in that, The first decellularization treatment stage includes the following steps: placing the biofilm material in a hypertonic solution and a hypotonic solution in sequence for decellularization treatment.

14. The method for preparing the tissue repair material according to claim 13, characterized in that, The first decellularization treatment stage meets one or more of the following conditions: 1) The hypertonic solution includes a 0.01M to 1M sodium chloride solution with added 0.01M to 1M alkali or acid; 2) The hypotonic solution includes water; 3) The first decellularization treatment step is repeated 2 to 5 times; and, 4) The process is carried out under shaking conditions, with the shaking time in the hypertonic solution and the hypotonic solution being 30 min to 90 min each independently.

15. The method for preparing the tissue repair material according to claim 14, characterized in that, The alkali in the hypertonic solution includes one or more of sodium hydroxide and potassium hydroxide.

16. The method for preparing the tissue repair material according to claim 14, characterized in that, The acid in the hypertonic solution includes one or more of hydrochloric acid and acetic acid.

17. The method for preparing the tissue repair material according to any one of claims 9 to 16, characterized in that, The biofilm material is treated as follows: removing attached fat, connective tissue and damaged edge tissue, defatting and inactivating viruses.

18. The method for preparing the tissue repair material according to claim 17, characterized in that, Degreasing meets one or more of the following conditions: (I) The degreasing agents used include one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane, and ethyl acetate; and, (II) The process is carried out under shaking conditions, with 2 to 4 degreasing treatments, each lasting 2 to 10 hours. After each degreasing treatment, fresh degreasing reagent is used before the next degreasing treatment.

19. The method for preparing the tissue repair material according to claim 17, characterized in that, The virus was inactivated using chemical methods.

20. The method for preparing the tissue repair material according to claim 19, characterized in that, The chemical method satisfies one or more of the following conditions: I) The inactivation reagents used include one or more of acids, bases, and alcohols, as well as... (ii) The inactivation of the virus is carried out under static conditions for 1 to 3 hours.

21. The method for preparing the tissue repair material according to claim 20, characterized in that, The alkali in the inactivation reagent includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide, and ammonia water.

22. The method for preparing the tissue repair material according to claim 20, characterized in that, The acid in the inactivating agent includes one or more of hydrochloric acid, nitric acid, phosphoric acid, and acetic acid; the alcohol in the inactivating agent includes one or more of ethanol, propanol, isopropanol, and methanol.

23. The method for preparing the tissue repair material according to any one of claims 9 to 16 and 18 to 22, characterized in that, After decellularization, the resulting tissue repair material is frozen, sectioned, and sterilized.

24. The method for preparing the tissue repair material according to claim 23, characterized in that, The freezing conditions include: freeze drying is carried out by cooling to -80℃ to -20℃ at a rate of 5℃ / min to 12℃ / min, and maintaining at -20℃ to -10℃ for 10h to 16h.

25. The method for preparing the tissue repair material according to claim 23, characterized in that, The thickness of the sliced ​​membrane is controlled to be 0.1 mm to 1 mm.

26. The method for preparing the tissue repair material according to claim 23, characterized in that, Sterilization can be performed using physical sterilization methods or chemical sterilization methods.

27. The method for preparing the tissue repair material according to any one of claims 9 to 16 and 18 to 22, characterized in that, The biofilm material satisfies one or more of the following conditions: (i) The biomembrane material is derived from the dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valves, small intestine, or basement membrane; and, (ii) The biofilm material is derived from pigs, cattle or sheep.

28. A tissue repair material, characterized in that, Prepared by the method for preparing tissue repair material according to any one of claims 9 to 27.

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